Method for determining the binding behavior of ligands which specifically bind to target molecules

ABSTRACT

The invention relates to a method for determining the binding behavior of ligands which specifically bind to target molecules at at least one binding site, whereby the markers are present in a native form and the determination of the concentrations K4 and K5 or of the quantities M2 and M1 is carried out by means of mass spectrometry.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Stage of InternationalApplication No. PCT/EP02/05543, filed May 21, 2002, which claimspriority under 35 U.S.C. § 119 of German Patent Application No. 101 25258.7, filed May 23, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a method for determining the binding behaviorof ligands which specifically bind to target molecules at at least onebinding site.

2. Discussion of Background Information

DE 198 14 775 A1 discloses a method for determining the bindingconstants of dissolved agents and substances on surfaces constituted byamphiphilic molecules. Therein, layers of amphiphilic molecules arebonded to a solid carrier so that a solid-supported membrane is formed.A defined quantity of this solid-supported membrane is contacted with amobile phase. The mobile phase contains a defined quantity of substanceswhich are to be examined with respect to lipid binding. After anincubation, the solid-supported membranes are separated from the mobilephase. The concentrations of the substances in the mobile phase or thesolid-supported membrane are determined. The method can be carried outwith solid-supported membranes and substances that are present indifferent quantity ratios. From the determined concentrations, thebinding constant can be calculated.

From WO 99/50669 A1 there is known a method for screening a compoundlibrary to determine the relative affinity of a plurality of putativeligands to a target receptor in relation to an indicator compound bymeans of frontal chromatography. Therein, the target receptors areimmobilized on the solid phase of a chromatographic column. The compoundlibrary is applied onto the chromatographic column and equilibratedtherewith at least partially. Subsequently, the indicator compound isapplied and its break through time, i.e., the time until it appears inthe effluent of the chromatographic column, is determined by means ofmass spectrometry. The break through time of the indicator compound isaffected by its affinity, the affinity of ligands present to the targetreceptor, and the number of target receptors which are not occupied bythe ligands. From the change in the break through time in comparison tothe known break through time of the indicator compound, it can bedetermined whether compounds of the compound library have an affinity tothe target receptors, and whether this affinity is higher or lower thanthat of the indicator compound.

A disadvantage of this method is that the target receptors areinvariably immobilized. They are not present in the native state,whereby it is possible to only a limited extent to say something aboutthe binding behavior on native target receptors. A further disadvantageof the method is that in kinetic studies, it leads to incorrect resultsregarding the binding behavior between ligands and the indicatorcompound. If a ligand and the indicator compound had the same affinitiesto the target receptor, the affinity of the ligand would be determinedas too low if the ligand shows slower binding kinetics relative to theindicator compound. A further disadvantage is that the quantity of theindicator compound which is bound or unbound, respectively, in thebinding equilibrium state cannot be determined.

WO 01/22078 A1 relates to a method for the identification of an activechemical substance from a mixture of active and inactive chemicalsubstances, where a target substance is added to the mixture. Complexesformed of the active substance and the target substance are separatedfrom the mixture. For identification, the active chemical substance maybe released from the complexes and identified by, for example, massspectrometry. Alternatively, the active substance may also be identifiedby comparing a chromatogram or a mass spectrum, respectively, of each ofthe mixture of active and inactive substances and the mixture afterseparation of the complex. A disadvantage thereof is that it must bepossible to determine each of the substances of the mixture.

WO 97/43301 A2 relates to a method for characterizing the members of acombinatorial library which bind to a certain domain. Therein, thedomain is mixed with the members of the combinatorial library to permita binding of the members to the domain. The formed complexes areseparated from the unbound members. The bound members are eluted fromthe complexes and analyzed by mass spectrometry. Therein, it is possibleto elute more weakly bound members separately from more strongly boundmembers. A disadvantage thereof is that each of the members of themixture must be determinable by mass spectrometry.

From WO 96/22530 A1 and U.S. Pat. No. 5,891,742 A there is known amethod for determining a compound which binds to a target compound froma mixture of compounds. Therein, the target compound is contacted withthe mixture of compounds. Unbound compounds are separated from formedcomplexes of the compounds with the target molecules. The formedcomplexes are analyzed by mass spectrometry. It is of disadvantagetherein that all compounds and complexes, respectively, of the bindingassay must be determinable by mass spectrometry.

From WO 00/00823 A1 there is known a method for identifying a ligandwhich covalently binds to a target molecule. In this method, a targetmolecule is contacted with several compounds of a substance library. Thecompound which forms a covalent bond with a chemically reactive group ofthe target molecule is identified by mass spectrometry. For thispurpose, the conjugate formed by the covalent bond may be fragmented.The bound compound may as well be released by cleaving a disulfide bond.For identification, the mass spectroscopic characteristics of thecompounds to be determined or of the fragment to be determined,respectively, must be known in this method.

From WO 99/45150 A1 there is known a method for determining the relativebinding affinity to a target substance of compounds of a combinatorialmixture. Therein, a first complex of the target substance and a standardcompound binding thereto is contacted with the combinatorial mixture ofcompounds, whereby second complexes are formed from these compounds andthe target substance. The mixture is analyzed by mass spectrometry. Fromthe quantity of first and second complexes contained therein, therelative binding affinity can be determined. Also in this assay, it mustbe possible to analyze all compounds which potentially form complexeswith the target molecules, and the complexes formed therefrom,respectively.

From WO 00/47999 A1 there is known a method for screening complexbiological samples for a ligand which binds to a target protein.Therein, the target protein is mixed with the complex biological sampleand incubated under conditions which permit the formation of complexesof ligands with the target protein. Formed ligand-target proteincomplexes are separated and caused to dissociate. Released ligands canbe analyzed by mass spectrometry following the separation of the targetprotein. The separation is effected in each case by means of sizeexclusion processes. Furthermore, WO 00/47999 A1 discloses the use of areference substance and of a known competitive ligand. The competitiveligand serves to determine whether a ligand binds specifically ornon-specifically to the selected target protein. For this purpose, thecompetitive ligand is mixed with the complex biological sample, theprocess is carried out as described, and it is determined whether themass spectroscopic signal of the ligand is affected by the presence ofthe competitive ligand. The method discloses a comparison of the bindingassays in the presence and absence of the competitive ligand. However,all bound ligands are invariably determined by mass spectrometry.

Moreover, it is generally known form the prior art to indirectlydetermine the binding of unlabeled ligands to a target molecule by meansof ligands which are labeled with a marker substance. Therein, theunlabeled ligands are incubated with the target molecules together witha defined portion of labeled ligands. The subsequent determination ofthe quantity of the marker substance which is bound to the targetmolecules through the labeled ligands allows the determination of thebinding behavior of the ligands which are bound to the target molecules.

The methods known in the prior art make it necessary to directly orindirectly quantify either the bound or the unbound ligands. With directquantification, this may involve a substantial expenditure, depending onthe ligand. For quantification, the employed detection system must becalibrated for each of the ligands to be quantified. This is necessaryalso for ligands of which it is not known whether they show an affinityto the target molecules. The indirect quantification by means of labeledligands involves substantial expenditure as well. Initially it isnecessary to label the ligands with a marker substance so that thelabeled ligands still have sufficient affinity to the target molecules.Frequently, various labeling methods with various marker substances mustbe tested for this purpose. Often, only radioactive marker substancesprove to be suitable. Handling these marker substances and the ligandslabeled therewith is dangerous, and because of the required safetyprecautions, it involves substantial expenditure. Furthermore, not everyligand is suitable for labeling by a certain desired method. This maynecessitate a search for suitable ligands.

Object of the present invention is the elimination of the disadvantagesof the prior art. In particular, there is to be provided a method whichmakes it possible to determine, with little expenditure, the specificbinding behavior of any ligands to any desired target molecules,unaffected to a large extent by the kinetics of the binding of theligands to the target molecules.

This problem is solved by the features of the claimed invention.

SUMMARY OF THE INVENTION

The present invention provides a method for determining the bindingbehavior of ligands which bind specifically to target molecules at atleast one binding site, which method comprises:

-   (A) preparing a first mixed phase wherein the ligands are contacted    with (i) markers which bind specifically to the target molecules,    and (ii) with the target molecules, wherein the ligands are present    in a concentration K1, the markers are present in a concentration K2    and the target molecules are present in a concentration K3;-   (B) preparing a second mixed phase wherein the markers are contacted    with the target molecules;-   (C) incubating the first and second mixed phases under identical    conditions, which conditions permit a binding of the ligands and the    markers to the target molecules, resulting in (a) a quantity M1 of    markers GM1 that are bound to target molecules and a concentration    K4 of unbound markers in the first mixed phase and (b) a quantity M2    of markers GM2 that are bound to target molecules and a    concentration K5 of unbound markers in the second mixed phase;-   (D) separating the markers GM1 from the first mixed phase and    separating the markers GM2 from the second mixed phase;-   (E1) determination of the concentrations K4 and K5, and calculating    a concentration K6 from K5, wherein K6 is a concentration of unbound    markers in the second mixed phase under the assumption that the    markers have been contacted therein in the concentration K2 with the    target molecules in the concentration K3, and determining the    binding behavior of the ligands from the ratio of the concentrations    K6 and K4, or-   (E2) determination of the quantities M1 and M2, and calculating a    quantity M3 from M2, wherein M3 is a quantity of bound markers in    the second mixed phase under the assumption that the markers have    been contacted therein in the concentration K2 with the target    molecules in the concentration K3, and determining the binding    behavior of the ligands from the ratio of the quantities M3 and M1.

In the above method, the markers are present in a native form and thedetermination of K4 and K5 or M1 and M2 is carried out by massspectrometry.

In one aspect of the method, in (B) the markers may be present in aconcentration K2 and the target molecules may be present in aconcentration K3.

In another aspect of the method, the markers GM1 may comprisespecifically bound markers SGM1 and the markers GM2 may comprisespecifically bound markers SGM2, and SGM1 and SGM2 may be liberatedafter (D), and the quantities thereof may be used in (E2) instead of M1and M2.

In yet another aspect, the method may further comprise repeating (A) to(E1) or (A) to (E2) wherein in at least one of (A) and (B) an agentwhich competes with the markers for a specific binding to the targetmolecules is added to at least one of the first and second mixed phasesin an amount which substantially prevents or cancels a specific bindingof the markers to the target molecules.

In another aspect, the method may further comprise repeating (A) to (E1)or (A) to (E2) at least once with a different ratio of K 1 and K3.

In a still further aspect of the method of the present invention, (D)may comprise one or more of dialysis, precipitation, adsorption, bindingto immobilized molecules which have an affinity to at least one of thetarget molecules, complexes of the target molecules and the markers andcomplexes of the target molecules and the ligands, centrifugation,chromatography, and filtration, preferably at least ultrafiltration.

In another aspect of the method, (D) may be carried out under conditionsunder which at least one of (a) the concentrations of the unboundmarkers in the first and the second mixed phases and (b) the amounts ofthe bound markers GM1 and GM2 remain constant.

In yet another aspect of the method, the mass spectrometry may becarried out in one of the MS, MS/MS and MS^(n) modes.

In another aspect, the mass spectrometry may be preceded by at least acapillary electrophoresis, a gas chromatography and/or a liquidchromatography. Preferably, the mass spectrometry is preceded by aFIPLC. The HPLC may comprise reverse phase chromatography.

In another aspect of the method, a sample preparation by means of atleast one of extraction, filtration, gas chromatography, capillaryelectrophoresis, liquid chromatography, FPLC®, reverse phasechromatography, size exclusion chromatography and affinitychromatography may be carried out prior to the mass spectrometry. Forexample, prior to the mass spectrometry a sample preparation by means ofat least one of solid phase extraction, liquid-liquid extraction,ultrafiltration and HPLC may be carried out.

In a still further aspect of the present method, the first and thesecond mixed phases of (A) and (B) may comprise more than 10 differenttarget molecules and/or more than 10 different markers, for example,more than 100 different target molecules and/or more than 100 differentmarkers. For example, the different markers may differ from each otherwith respect to one or more of the molar mass, the formation of ions ordaughter ions in the mass spectrometry, the chromatographic behavior andthe electrophoretic behavior.

In yet another aspect, the first and the second mixed phases of (A) and(B) may comprise a common marker which is specific to different targetmolecules.

In another aspect, the first mixed phase of (A) may comprises more than10 different target molecules and/or more than 10 different markers, forexample, more than 100 different target molecules and/or more than 100different markers.

In another aspect of the method, the ligands may have been generated bymethods of combinatorial chemistry.

In another aspect, the method may further comprise identifying theligands which are bound by the target molecules and/or determining thequantity of ligands which are bound by the target molecules. Forexample, mass spectrometry may be used for identifying the bound ligandsand/or for determining the quantity thereof.

In yet another aspect of the method, prior to mass spectrometry thebound ligands may be specifically liberated from the target molecules.

In a still further aspect, the markers may be used as external standardfor the determination of K4and K5 or M1 and M2.

In another aspect of the method, after (D) at least one marker whichcomprises a marking substance may be added as an internal standard tothe first mixed phase and/or the second mixed phase.

In yet another aspect, the target molecules may comprise targetmolecules that are embedded in membrane structures and/or the targetmolecules may comprise target molecules that are not bound to a solidsupport.

In another aspect, the method may further comprise a regeneration of thetarget molecules after one of (D) and (F2).

In a still further aspect, the target molecules, the markers and/or theligands may be selected from peptides, proteins, hormones, nucleicacids, sugars, polymers, low molecular weight compounds of natural orsynthetic origin and structures on or inside cells, cell fragments, cellhomogenates, synaptosomes, liposomes, vesicles, tissue cuts, viruses,components and fragments thereof, capsides, components and fragmentsthereof, and mixtures of natural substances. For example, at least oneof the target molecules, markers and ligands may be selected fromreceptors, prions, enzymes, transport proteins, ion channels,antibodies, synaptic plasma membrane vesicles and vesicles found insidecells.

The present invention also provides a process for determining thecapability of a chemical species to exhibit pharmaceutical activity.This process comprises carrying out the method of the present invention,including the various aspects thereof.

In one aspect of this process, the ligands may comprise the chemicalspecies.

The present invention also provides a new pharmaceutically activespecies whose capability to exhibit a pharmaceutical activity has beendetermined by a process which comprises using the method of the presentinvention, including the various aspects thereof.

-   (a) preparation of a first mixed phase wherein the ligands in a    concentration K1 are contacted with markers, in a concentration K2,    which specifically bind to the target molecules, and with the target    molecules in a concentration K3, and of a second mixed phase wherein    the markers are contacted with the target molecules, wherein after    the contacting, the first and second mixed phases are incubated    under identical conditions which permit a binding of the ligands and    the markers to the target molecules,-   (b) separation of the bound markers GM1 contained in the first mixed    phase from the first mixed phase, and of the bound markers GM2    contained in the second mixed phase from the second mixed phase, and-   (c1) determination of a concentration K4 of the unbound markers in    the first mixed phase, and of a concentration K5 of the unbound    markers in the second mixed phase, wherein a concentration K6 is    determined from the concentration K5, which concentration K6    corresponds to the concentration of the unbound markers in the    second mixed phase under the assumption that the markers in the    concentration K2 have been contacted therein with the target    molecules in the concentration K3, and determination of the binding    behavior of the ligands from the ratio between the concentrations K6    and K4, or-   (c2) determination of an quantity M1 of the bound markers GM1 and of    a quantity M2 of the bound markers GM2, wherein a quantity M3 is    determined form the quantity M2, which quantity M3 corresponds to    the quantity of the bound markers in the second mixed phase under    the assumption that the markers in the concentration K2 have been    contacted therein with the target molecules in the concentration K3,    and determination of the binding behavior of the ligands from the    ratio of the quantities M3 and M1,    wherein the markers are present in a native form, and the    determination of the concentrations K4 and K5 or of the quantities    M2 and M1 is effected by means of mass spectrometry.

The binding behavior is to be construed to mean any behavior whichcharacterizes the specific binding of the ligands to the targetmolecules at at least one binding site. It may be a relative bindingbehavior. In this case, the binding behavior indicates whether theligands bind better or worse than the markers to the target molecules,and optionally, to which extent they bind thereto. It may as well be anabsolute binding behavior. In this case, the affinity of the ligands tothe target molecules, or the quantity of ligands which are bound to thetarget molecules are determined. The determination of the bindingbehavior may as well consist of a mere approximation of the bindingbehavior.

The term mixed phase comprises both a pure solution and a suspension.The mixed phase may contain membrane structures such as, e.g., vesicles.The target molecules may be present in solution, suspension, immobilizedor embedded in membrane structures, in particular, vesicles. Further,instead of the target molecules, the ligands may be immobilized. Thetarget molecules or the ligands may be present immobilized on, e.g., thewall of a vessel or on particles. Further, in the case of targetmolecules which are embedded in vesicles, the vesicles may beimmobilized, e.g., by binding to a solid phase. The conditions accordingto lit. a are selected so that a binding of the ligands and the markersto the target molecules can take place, in particular, as regardstemperature and duration of the incubation. The incubation may becarried out until a binding equilibrium is at least almost reached.

Markers are understood to be known, quantifiable agents which at theemployed concentrations K2 and K3 and under the conditions according tolit. a, bind specifically to the target molecules. The binding of themarkers to the target molecules is inhibited by the binding of theligands. This may occur due to, e.g., an allosteric inhibition of thebinding, or because the markers and the ligands compete for the bindingto the binding site of the ligands on the target molecules. Theconcentration K6 or the quantity M3, respectively, may be determined bycalculation or graphically from the binding properties of the markersand the concentration K5 or the quantity M2, respectively. Theconcentration K6 is that concentration of unbound marker, and thequantity M3 is that quantity of bound marker, which would be present inthe second mixed phase after the incubation if the markers at theconcentration K2 had been contacted therein with the target molecules atthe concentration K3. From the ratio between the concentrations K6 andK4 or the quantities M3 and M1, respectively, the quantity of themarkers whose binding to the target molecules is inhibited, andtherefrom the binding behavior of the ligands, may be determined. Theratio in the sense of the invention may be both a relation and adifference. Both the markers and the ligands and the target moleculesmay be of a uniform type, or may differ from each other.

The separation according to lit. b may take place, for example, bycentrifugation of formed complexes. The complexes may contain targetmolecules and ligands, target molecules and markers, or targetmolecules, ligands and markers. It is to be understood that instead of aconcentration, a quantity, and instead of a quantity, a concentrationmay be determined.

“Native form” means that the markers do not have any modifications whichare necessary for the detection, quantification or identification. Suchmodifications may, for example, consist in a labeling with a markersubstance.

The advantage of the method is that the influence of kinetic differencesin the binding behavior between the ligands and the markers on theresult can to a large extent be eliminated in that incubation isconducted until a binding equilibrium is at least almost reached. Afurther advantage of the method according to the present invention isthat the target molecules may be present in a native, in particular, notimmobilized form. Thereby the method permits to say something about thenative binding behavior.

A further advantage of the method is that in the case of ligands whichare difficult to quantify directly, only the markers need to bequantified. Thus the method is not subject to limitations which rest inthe nature of the ligands and cause problems in the quantification. Withrespect to the markers, there is the advantage that a development ofmarkers from agents which bind specifically to the target molecules iseliminated, because there is no need for the markers to be labeled. Thusa search for agents which are suitable for labeling by a particularlabeling method and bind specifically to the target molecules is notnecessary. By the same token, there is no need for a search for agentswhich bind specifically to the target molecules and which still have asufficient affinity after the labeling. Moreover, the expenditure whichis necessary due to the handling of the utilized marker substances iseliminated, which expenditure may be quite high, in particular, withradioactive marker substances. A health hazard associated with theemployment of radioactive marker substances is not present, either.

The use of mass spectrometry is advantageous in that markers which arepresent in a native form can be detected, quantified and, when differentmarkers are used at the same time, identified.

In the method according to the present invention only the affinity ofthe markers, and not the affinity of the ligands to be studied, to thetarget molecules is of relevance. If the markers have a sufficientaffinity to the target molecules, the binding behavior of ligands whichhave very low affinities to the target molecules may be determined byselecting a correspondingly high concentration K1. The utilized markersmay be selected so that they can still be detected in low concentrationsas well. A further advantage of the method is that an unspecific bindingof the ligands to be studied has almost no impact on the result of themeasurement. Substantially only the non-specific binding of the markersmust be taken into account.

In an advantageous embodiment, in step lit. a the markers in the secondmixed phase are contacted in a concentration K2 with the targetmolecules in a concentration K3. Thereby, the determination of theconcentration K6 in step lit. c1 or of the quantity M3 in step lit. c2is simplified, because under these conditions the concentration K6 isthe same as the concentration K5, and the quantity M3 is the same as thequantity M2.

In a further advantageous embodiment, after step lit. b the specificallybound markers SGM1 among the bound markers GM1 and the specificallybound markers SGM2 among the bound markers GM2 are released after thestep lit. b. In step lit. c2, the released specifically bound markersSGM1 take the place of the bound markers GM1, and the releasedspecifically bound markers SGM2 take the place of the bound markers GM2.The release may be effected, for example, by adding an excess ofmolecules which specifically inhibit the binding of the markers. Suchmolecules may, for example, be competitors which displace thespecifically bound markers from the binding site. Another possibilityconsists in removing by a washing step non-specifically bound markersfrom the target molecules so that only specifically bound markers remainbonded. The specifically bound markers may then be released by means ofnon-specific methods. By releasing the specifically bound markers theproportion of non-specifically bound markers, can be taken into account.

Further, for taking into account a non-specific binding of the markersto the target molecules the steps lit. a to c1 or c2 may be carried outan additional time, wherein in step lit. a a material which competeswith the markers for the specific binding to the target molecules isadded to the first and/or the second mixed phase in such an amount thata specific binding of the markers to the target molecules is largelyprevented or terminated. The concentration K4 determined thereby insteps lit. c1 or c2 is identified as K4′, K5 as K5′ and K6 as K6′, andthe quantity M1 as M1′, M2 as M2′ and M3 as M3′. The values determinedwithout the agent are still identified as K4, K5, K6, M1, M2, and M3.The difference between K4′ and K4, K5′ and K5, and K6′ and K6 in eachcase is the concentration of the specifically bound markers. M1′, M2′and M3′ each are the quantities of the non-specifically bound markers.The difference between M1 and M1′, M2 and M2′ and M3 and M3′ is thequantity of the specifically bound markers.

In a preferred embodiment of the invention, for the determination of theaffinity the steps lit. a to c1 or c2 are carried out at least twicewith a different ratio of the concentrations K1 to K3. Preferably, inthis case the incubation according to step lit. a is carried out untilthe binding equilibrium is at least almost reached. From the quantitiesof the markers whose binding has been inhibited at the different ratioof the concentrations, the affinity can be determined. This can be doneby means of a non-linear regression analysis of the competition ordisplacement graph. The competition or displacement graph represents theinhibition of the binding of the markers by ligands as a function of theconcentration of unbound ligands.

Preferably, the separation according lit. b is effected by dialysis,precipitation, adsorption, binding to immobilized molecules which havean affinity to the target molecules or to formed complexes of the targetmolecules and the markers and/or the ligands, centrifugation,chromatography, or filtration, in particular, ultrafiltration. Theseparation according to lit b. is preferably carried out underconditions under which the concentration of the unbound markers in thefirst and in the second mixed phases and/or the quantity of the boundmarkers GM1 and GM2 remains constant. Any method wherein the separationof bound ligands is carried out under conditions under which theproportions of the bound and the unbound markers and ligands remainconstant is suitable for this purpose. As such a method, the separationof formed complexes of target molecules and markers and/or ligands underbinding conditions, e.g., by means of centrifugation or filtration, maybe contemplated. The advantage of the separation under the mentionedconditions is that a cancellation of a binding equilibrium which iscaused by the separation, for example in a washing step, is avoided. Adissociation of the complexes which are formed from the target moleculesand the markers and/or the ligands which is caused by the cancellationof the binding equilibrium does not take place. An error in thequantification of the bound ligands resulting therefrom is avoided. Afurther advantage of the separation under the mentioned conditions isthat the first and the second mixed phases do not have to be separatedfrom the bound markers GM1 and GM2 quantitatively. It is sufficient toseparate a defined portion of each of the first and the second mixedphases. From this portion and the concentrations K4 and K5 determined instep lit. c1 the amount of marker whose binding has been inhibited bythe ligands may be determined.

Advantageously, the mass spectrometry is carried out in the MS, MS/MS orMS^(n) mode. In the MS mode, ions formed by the analyte in the ionsource of a mass spectrometer, in the MS/MS mode selectively daughterions of the ions formed by the analyte, and in the MS^(n) modeselectively further daughter ions of the ions formed by the analyte arequantified. Mass spectrometry may be preceded by a capillaryelectrophoresis or a gas or liquid chromatography, preferably HPLC, inparticular, by using reverse phase chromatography. A separation ofmolecules which can be distinguished in the mass spectrometry only withdifficulty can be achieved thereby. The molecules can then separately beanalyzed in the mass spectrometry.

Preferably, prior to the mass spectrometry a sample preparation by meansof extraction, preferably solid phase or liquid-liquid extraction,filtration, in particular, ultrafiltration, gas chromatography,capillary electrophoresis, liquid chromatography, preferably HPLC,FPLC®, reverse phase chromatography, size exclusion chromatographyand/or affinity chromatography is carried out. Sample preparation meansthe work-up of a sample from which the concentrations K4 and K5 or thequantities M2 and M1 are to be determined. This may be necessary, forexample, if certain components must be removed from the sample. Thesemay be components which are harmful to a mass spectrometer, for example,because they would plug or attack the feed lines, or which reduce thesensitivity of a mass spectrometric measurement, e.g., because theyreduce the signal of the marker to be analyzed. Furthermore, these maybe components which would interfere with a method which precedes themass spectrometry.

The first and the second mixed phases according to lit. a may preferablycontain more than 10, in particular, more than 100 various targetmolecules and/or markers. In a preferred embodiment the first and thesecond mixed phase according to lit. a contain a common marker which isspecific to various target molecules. If various markers are used, eachmarker specifically binds to at least one binding site of apredetermined target molecule. Preferably, the various markers differfrom each other at least as regards the molar mass, the formation ofions or daughter ions in the mass spectrometry, or the chromatographicor electrophoretic behavior. Advantageously, the first mixed phaseaccording to lit. a preferably contains more than 10, in particular,more than 100 different ligands. The ligands may have been generated bymethods of combinatorial chemistry. Such methods are known, for example,from Felder, E. R., Chimia 48 (1994), 521–541, Gallop et al., J. Med.Chem. 37 (1994), 1233–1251, Houghten, R. A., Trends Genet. 9 (1993),235–239, Houghten et al., Nature 354 (1991), 84–86, Lam et al., Nature354 (1991), 82–84, Carell et al., Chem. Biol. 3 (1995), 171–183, Maddenet al., Perspectives in Drug Discovery and Design 2 (1994), 269–285,Cwirla et al., Biochemistry 87 (1990), 6378–6382, Brenner, S. andLerner, R. A., Proc. Natl. Acad. Sci. USA 89 (1992), 5381–5383, Gordonet al., J. Medicinal Chemistry 37 (1994), 1385–1401 and Lebl et al.,Biopolymers 37 (1995), 177–198.

By using several different ligands, target molecules and/or markers, theefficiency of the method can be increased significantly. When the methodis carried out with different markers, only each of the markers has tobe quantified in order to determine if ligands which have an affinity tothe target molecules were present in the first mixed phase. Thus themethod is suitable for high throughput screening. To find out which ofthe ligands have an affinity to the target molecules, a deconvolutionstrategy may be pursued. Here, the method is carried out anew with areduced number or a different combination of ligands to be studied. Bycomparing the results it can then be determined which of the ligands hasbonded to the target molecules in which amount. Also, the differentmarkers may have different graduated affinities to the target molecules.By determining or approximating the amount of those markers whosebinding to the target molecules has been inhibited, the affinity of theligands to the target molecules may be determined or at leastapproximated.

In one embodiment, following step lit. c1 or c2 the bound ligands areidentified by mass spectrometry and/or the amount of bound ligands isdetermined by mass spectrometry. For this, the bound ligands may bespecifically released prior to mass spectrometry, for example, by theaddition of specific competitors. Such a method is very efficient in ahigh throughput screening in which many substances which predominantlydo not bind to the target molecules are studied at the same time withrespect to their binding behavior. The identification of the boundligands or the determination of the quantity of the bound ligands,respectively is necessary only if it results from step lit. c1 or c2that bound ligands are present at all.

For the determination of the concentrations K4 and K5 or of thequantities M1 and M2 the markers may be used as external standard. Also,it is possible to add to the first and/or the second mixed phase afterstep lit. b as internal standard at least one marker which has a markingsubstance. Such an internal standard facilitates the determination ofthe concentrations K4 and K5 because it is affected to the same extentas the marker itself. Thus, losses in the sample preparation or apossibly necessary concentration of the sample may be taken into accountas readily as interfering factors which affect the mass spectrometricdetermination.

The target molecules may be present embedded in membrane structures.This may be advantageous, for example, with target molecules which arenaturally present embedded in membrane structures, and whose bindingproperties are altered when they are not embedded in membranestructures. In an advantageous embodiment, the target molecules are notbound to a solid support. Thereby a change in the binding behavior ofthe target molecules by the binding to a solid support and and/or aninfluence on the kinetics of the binding are avoided.

It is possible to regenerate the target molecules after the steps lit. bor c2 have been carried out. This permits a reuse thereof. This makessense, in particular, with valuable target molecules, e.g., targetmolecules on cells on which several studies are to be conducted. Thetarget molecules, markers or ligands may be selected from the followinggroup: peptides, proteins, in particular, receptors, prions, enzymes,transport proteins, ion channels or antibodies, hormones, nucleic acids,sugars, polymers, low molecular weight compounds of natural or syntheticorigin and structures on or inside cells, cell fragments, cellhomogenates, synaptosomes, liposomes, vesicles, in particular, synapticplasma membrane vesicles and vesicles which are found in cells, tissuecuts, viruses, their components or fragments of these components,capsides, their components or fragments of these components and mixturesof natural substances.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following, the invention is explained in greater detail by thedrawing and by means of working examples. There are shown in

FIG. 1 The result of a method carried out according to the presentinvention, wherein there were employed μ-opioid receptors as targetmolecules, morphine as a marker and naloxone as a ligand in differentconcentrations, and

FIG. 2 The result of a control experiment wherein there were employedμ-opioid receptors as target molecules, [³H]DAMGO as a marker andnaloxone as a ligand.

DETAILED DESCRIPTION OF THE INVENTION

In all working examples, human μ-opioid receptors served as targetmolecules in the binding assays. The target molecules were provided bymeans of a membrane preparation of transfected human μ-opioid receptorswhich express CHO-K1 cells of the company Biotrend, Cologne.

WORKING EXAMPLE 1 Screening of Substance Libraries by Determination ofan Unbound Marker

1.1. Binding Assays

An assay B0 contained the μ-opioid receptors in a concentration of 5.5nmol/L, 5 mmol/L of MgCl₂ and 50 mmol/L of Tris HCl, pH 7.4, in a totalvolume of 250 μL. The assay B0 was incubated in a 1.5 ml PP reactionvessel at 25° C. for 150 minutes. Subsequently, the membrane particleswere centrifuged off at 50,000 g at 4° C. for 20 minutes. 200 μL weretaken from the resultant supernatant Ü0. The following binding assayswere treated like B0:

-   -   A binding assay B1 which was composed like binding assay B0 and        additionally contained 10 nmol/L of morphine as a marker.    -   A binding assay B2 which was composed like binding assay B1 and        additionally contained (±)-methadone in a concentration of 50        μmol/L.    -   A binding assay B3 which was composed like binding assay B1 and        additionally contained the substances of a library 1, consisting        of 4-chloroaniline, 2,3-dichloroaniline, methyl benzoate,        acetoanilide, amitryptiline, (+)-bicuculine, phenol, naloxone,        benzoic acid and tramadol, each in a concentration of 1 μmol/L.    -   A binding assay B4 which was composed like binding assay B1 and        additionally contained the substances of a library 2, each in        concentration of 1 μmol/L. The library 2 consists, except for        naloxone, of the substances of the library 1.        From B1–B4 supernatants Ü1–Ü4 were recovered.        1.2. Quantification of the Marker

For quantification, a mass spectrometric determination was carried outunder the following conditions on an Applied Biosystems API 2000 withTurbolonSpray source in the MRM mode to which a Hewlett Packard HP1100LC system was coupled:

-   Column: Superspher 60 RP select B 4 μm 125×2 mm-   Eluent: 0.1% HCOOH in water/acetonitrile (80/20)-   Flow: 0.4 ml/minute (isocratic)-   Sample volume: 20 μL (injected by means of Autosampler)

Morphine was directly measured in the supernatants Ü1–Ü4 at the masstransitions 286.1/115.1 and 286.1/128.1. It was quantified in comparisonto morphine which was dissolved in Ü0 in concentrations of 2 nmol/L to10 nmol/L as external standard. With each sample two measurements werecarried out.

1.3. Analysis

In Ü1–Ü4 the following concentrations of unbound morphine weredetermined as average of two measurements:

-   Ü1: 2.25 nmol/L-   Ü2: 7.66 nmol/L-   Ü3: 7.92 nmol/L-   Ü4: 2.03 nmol/L

From each difference of the employed concentrations of morphine and theconcentrations of unbound morphine determined for Ü1–Ü4, the followingconcentrations of bound morphine were determined for B1–B4:

-   B1: 7.75 nmol/L-   B2: 2.34 nmol/L-   B3: 2.08 nmol/L-   B4: 7.97 nmol/L

In B1, the binding of morphine was determined without additionalcompetitor as total binding. By contrast, in B2 the binding of morphinewas determined in the presence of 50 μmol/L of methadone underconditions which could be expected to approximate a total occupation ofthe target molecules by methadone. Morphine has merely boundnon-specifically in this case. Since in B3 the concentration of boundmorphine is only of the order of magnitude of the non-specific binding,there is at least one substance in library 1 which has a significantaffinity to the target molecules at a concentration of 1 μmol/L. Sincein B4 the concentration of bound morphine is of the order of magnitudeof the total binding, there is no substance in library 2 which at aconcentration of 1 μmol/L, has a significant affinity to the targetmolecules. Since the libraries 1 and 2 differ only in that naloxone iscontained in library 1, but not in library 2, the affinity exhibitingsubstance can only be naloxone.

WORKING EXAMPLE 2 Determination of the Affinity of a Ligand byMeasurement of the Unbound Marker

2.1. Binding Assays

A first binding assay B1 contained the μ-opioid receptors in aconcentration of 5.5 nmol/L, 10 nmol/L of morphine as a marker, 5 mmol/Lof MgCl₂ and 50 mmol/L of Tris HCl, pH 7.4, in a total volume of 250 μL.The binding assay B1 was incubated in a 1.5 ml PP reaction vessel at 25°C. for 150 minutes. Subsequently, the membrane particles werecentrifuged off at 50,000 g at 4° C. for 20 minutes. 200 μL were takenfrom the resultant supernatant Ü1. The following binding assays weretreated like B1:

-   -   A second binding assay B2 which was composed like binding assay        B1 and additionally contained (±)-methadone in a concentration        of 50 μmol/L.    -   Five further binding assays B3–B7 which were composed like B1        and additionally contained naloxone in the following        concentrations: B3: 1 nmol/L of naloxone, B4: 10 nmol/L of        naloxone, B5: 30 nmol/L of naloxone, B6: 100 nmol/L of naloxone,        B7: 1 μmol/L of naloxone.

In a second test series which was carried out in an identical manner,the binding assays B1–B7 were substituted by the identical bindingassays B1*–B7*. From B1–B7 the supernatants Ü1–Ü7, and from B1*–B7* thesupernatants Ü1* –Ü7* were recovered.

2.2. Quantification of the Marker

The mass spectrometric determination was carried out as described under1.2. Additionally, naloxone was quantified at the mass transition328.1/310.1. With each sample two measurements were carried out.

2.3 Analysis

In Ü1–Ü7 and Ü1*–Ü7* the following concentrations of unbound morphineand naloxone were determined as the average of two measurements:

Morphine Naloxone Ü1: 2.25 nmol/L — Ü2: 7.66 nmol/L — Ü3: 2.78 nmol/L0.35 nmol/L Ü4: 4.15 nmol/L 4.54 nmol/L Ü5: 4.75 nmol/L 15.2 nmol/L Ü6:7.00 nmol/L 77.2 nmol/L Ü7: 8.23 nmol/L  858 nmol/L Ü1*: 2.24 nmol/L —Ü2*: 6.29 nmol/L — Ü3*: 2.18 nmol/L 0.14 nmol/L Ü4*: 3.16 nmol/L 2.96nmol/L Ü5*: 4.43 nmol/L 13.0 nmol/L Ü6*: 5.50 nmol/L 61.7 nmol/L Ü7*:5.86 nmol/L  668 nmol/L

From each difference of the employed concentrations of morphine and theconcentrations of unbound morphine determined for Ü1 –Ü7 and Ü1*–Ü7*,the following concentrations of bound morphine were determined for B1–B7and B1*–B7*:

B1: 7.75 nmol/L B1*: 7.76 nmol/L B2: 2.34 nmol/L B2*: 3.71 nmol/L B3:7.22 nmol/L B3*: 7.82 nmol/L B4: 5.85 nmol/L B4*: 6.84 nmol/L B5: 5.15nmol/L B5*: 5.57 nmol/L B6: 3.00 nmol/L B6*: 4.50 nmol/L B7: 1.77 nmol/LB7*: 4.14 nmol/L

In B1 and B1*, the binding of morphine was determined without additionalcompetitor as total binding. By contrast, in B2 and in B2*the binding ofmorphine was determined in the presence of 50 μmol/L of methadone underconditions which could be expected to approximate a total occupation ofthe target molecules by methadone. Morphine has merely boundnon-specifically in this case. The specific binding results from thedifference of the concentrations determined for B1 and B2 and B1*andB2*, respectively. In FIG. 1 the concentration of the bound morphine isrepresented as a function of the concentration of the unbound, i.e.,free naloxone.

By means of a non-linear regression analysis carried out with theprogram Prism 2.01 Graph Pad, San Diego, USA, the concentration of freenaloxone at which 50% of the morphine are specifically bound wasdetermined. This value is called IC₅₀ value. For the binding assaysB1–B7, an IC₅₀ value of 11.9 nmol/L, and for the binding assays B1*–B7*,an IC₅₀ value of 11.6 nmol/L was determined.

The K_(i) value as a measure of the affinity of naloxone to the targetmolecules may be determined as follows according to Hulme, E. C. andBirdsall, N. J. M., Strategy and tactics in receptor-binding studies inReceptor-Ligand-Interactions, ed. Hulme, E. C., Oxford UniversityPresss, Oxford (1992), 63–176, in binding experiments in which theconcentration of the free marker is different to a non-negligible extentfrom the starting concentration of the marker:

$K_{i} = \frac{I\; C_{50}}{{2\frac{( {L^{*} - L_{0}^{*}} )}{L_{0}^{*}}} + 1 + \frac{L^{*}}{K_{d}}}$The meanings are:

-   L*: Concentration of the marker at the IC₅₀ value.-   L₀*: Concentration of the free marker in the absence of the    competitor and-   K_(d): Affinity of the marker to the target molecules.

From Raynor, K., Kong, H., Mestek, A., Bye, L. S., Tian, M., Liu, J.,Yu, L., and Reisine, T., Characterisation of the cloned Mu opioidReceptor, J. Pharmacol. Exp. Ther. 272 (1995), 423–428, it is known thatthe K_(d) value for morphine is 2.0 nmol/L. From this, there results forB1–B7 at

-   IC₅₀=11.9 nmol/L-   L*=4.8 nmol/L-   L₀*=2.25 nmol/L    a K_(i) for naloxone of 1.4 nmol/L with respect to the human    μ-opioid receptors.

For B1 *–B7*, there results at

-   IC₅₀=11.6 nmol/L-   L*=4.2 nmol/L-   L₀*=2.24 nmol/L    a K_(i) for naloxone of 1.7 nmol/L with respect to the human    μ-opioid receptors.    2.4. Control Experiment

Competition Naloxone/[³H]DAMGO on Human μ-Opioid Receptors

[³H]DAMGO is the ³H labeled μ-opioid receptor agonistTyr-D-Ala-Gly-N-Methyl-Phe-Gly(o1)-encephaline. 22 μg of protein of amembrane preparation of transfected CHO K1 cells of the companyBiotrend, Cologne, which preparation contained 30 fmol of human μ-opioidreceptors, were incubated as described in Wanner, K. T., Praschak, I.,Höfner, G. and Beer, H., Asymmetric Synthesis and Enantiospecificity ofBinding of 2-(1,2,3,4-Tetrahydro-1-isoquinolinyl)-ethanol derivatives toμ- and κ-receptors, Arch. Pharm. 329 (1996), 11–12, with 4.5 nmol/L of[³H]DAMGO and 0 nmol/L, 1 nmol/L, 10 nmol/L, 30 nmol/L, 100 nmol/L and 1μmol/L of naloxone for 60 minutes at 25° C. in a total volume of 250 μL.The non-specific binding was determined in the presence of 50 μmol/L of(±)-methadone. Two test series were carried out in triplicate each. InFIG. 2 the concentration of the formed [³H]DAMGO is represented as afunction of the concentration of the naloxone employed. By means of anon-linear regression analysis carried out with the program Prism 2.01Graph Pad, San Diego, USA, the IC₅₀ value, i.e., the concentration ofnaloxone at which 50% of the [³H]DAMGO are specifically bound wasdetermined. From Raynor, K., Kong, H., Mestek, A., Bye, L. S., Tian, M.,Liu, J., Yu, L., and Reisine, T., Characterisation of the cloned Muopioid Receptor, J. Pharmacol. Exp. Ther. 272 (1995), 423–428, it isknown that the Kd value for DAMGO is 1.4 nmol/L. According to Cheng, Y.C. and Prusoff, W. H., Relationship between the inhibition constant Kiand the concentration of inhibitor which causes 50 cent inhibition IC50of an enzymatic reaction, Biochem. Pharmacol. 22 (1973), 3099–3108, thefollowing K_(i) values of naloxone with respect to human μ-opioidreceptors were calculated from the K_(d)-value for DAMGO and the IC₅₀values of 18 nmol/L and 17 nmol/L from the first and second test series,respectively:

-   1st Test series K_(i)=4.2 nmol/L-   2nd Test series K_(i)=4.1 nmol/L

WORKING EXAMPLE 3 Screening of Substance Libraries By Measuring theBound Marker After Its Controlled Release

3.1. Binding Assays

The binding assays B1–B4 described in Working Example 1 were used andtreated as described under 1.1. Pellets P1–P4 obtained from B1–B4 aftercentrifugation were treated as follows: P1–P4 were each washed threetimes with 200 μL of 5 mmol/L of MgCl₂ and 50 mmol/L of Tris HCl, pH7.4. Subsequently, P1–P4 were each resuspended in 200 μL of 5 mmol/L ofMgCl₂ and 50 mmol/L of Tris HCLl, pH 7.4, and incubated for 15 minutesat room temperature in a shaking water bath and centrifuged at 50,000×gfor 20 minutes at 4° C. The resultant supernatants were discarded andthe obtained pellets P1′–P4′ were each resuspended with 200 μL of 50μnmol/L of (±)-methadone in 5 mmol/L of MgCl₂ and 50 mmol/L of Tris HCl,pH 7.4 and once again incubated as before for 15 minutes. The assayswere subsequently centrifuged as before, whereby the supernatants F1–F4were recovered.

3.2. Quantification of the Marker

The morphine contained in F1–F4 was quantified as described under 1.2.

3.3. Analysis

In F1–F4 the following concentrations of unbound, i.e., releasedmorphine were measured as average of two measurements:

-   F1: 4.84 nmol/L-   F4: 4.80 nmol/L-   F2/F3: morphine not detectable

In B1, the binding of morphine was determined without additionalcompetitor as total binding. By contrast, in B2 the binding of morphinewas determined in the presence of 50 μmol/L of methadone underconditions which could be expected to approximate a total occupation ofthe target molecules by methadone. Morphine has merely boundnon-specifically in this case. From P2′ apparently no morphine could bereleased, since morphine is not detectable in F2. Accordingly, it can beassumed that the morphine which was detected in F1 and released from P1′was bound specifically. Since about as much morphine was released fromP4′ in F4 as from P1′ into F1, the library 2 apparently does not containa ligand with pronounced affinity to the target molecules. Apparently nomorphine could be released from P3′, because morphine was not detectablein F3. Accordingly, the library 1 contains at least one substance whichhas a significant affinity to the target molecules at a concentration of1 μmol/L. Since the libraries 1 and 2 differ only in that naloxone iscontained in library 1, but not in library 2, the substance withaffinity can only be naloxone.

WORKING EXAMPLE 4 Screening of Substance Libraries By Measuring theUnbound Marker and Subsequent Identification of Ligands Having AffinityFollowing Their Controlled Release

4.1. Binding Assays

With the exception of each containing the substances of the library 1and of the library 2 in a concentration of 10 nmol/L instead of 1μnmol/L, the binding assays B1#–B4# were the same as the binding assaysB1–B4 in Working Example 1. The binding assays were treated as describedunder 1.1. By centrifugation, supernatants Ü1#–Ü4# and pellets P1#–P4#were obtained from the binding assays B1#–B4#. P4# was discarded.P1#–P3# were each washed three times with 200 μL of 5 mmol/L of MgCl₂and 50 mmol/L of Tris HCl, pH 7.4. Subsequently, P1#–P3# were eachresuspended in 200 μL of 5 mmol/L of MgCl₂ and 50 mmol/L of Tris HCl, pH7.4, incubated for 15 minutes at room temperature in a shaking waterbath, and centrifuged at 50,000× g for 20 minutes at 4° C. The resultantsupernatants were discarded and the obtained pellets P1#′–P3#′were eachresuspended with 200 μL of 50 μmol/L of (±)-methadone in 5 mmol/L ofMgCl₂ and 50 mmol/L of Tris HCl, pH 7.4, and once again incubated asbefore for 15 minutes. The assays were subsequently centrifuged asbefore, whereby the supernatants F1#–F3# were recovered.

4.2 Quantification of the Ligands

Unbound or free morphine, respectively, was quantified in Ü1#–Ü4# and inF1#–F3# as described under 1.2. In F3#, also the individual substancesof the library 1 were additionally determined at the respective specificmass transitions. However, except for naloxone, none of the substancescould be detected in F3#. The released naloxone was quantified at themass transition 328.1/310.1. Two measurements were carried out with eachsample.

4.3. Analysis

In Ü1#–Ü4# the following concentrations of unbound morphine weredetermined as mean values:

-   Ü1#: 2.39 nmol/L-   Ü2#: 7.80 nmol/L-   Ü3#: 5.02 nmol/L-   Ü4#: 2.76 nmol/L

Since B4# from the library 2 apparently did not contain a ligand withsignificant affinity to the target molecules, P4# was discarded.

In F1#–F3# the following average concentrations of released morphine andnaloxone were determined:

Naloxone Morphine F1: 4.63 nmol/L F2: morphine not detectable F3: 2.30nmol/L 2.93 nmol/L

In F3#, naloxone and morphine could be detected in approximately thesame concentrations. Apparently, among the substances of library 1 onlynaloxone has high affinity to the target molecule, while the remainingsubstances have a distinctly lower affinity. Since the identified ligandnaloxone and the marker morphine were contained in binding assay B3# inthe same concentrations, it can be inferred that the released identifiedligand naloxone has an affinity to target molecules which is in therange of the affinity of the marker morphine.

WORKING EXAMPLE 5 Screening of Test Sustances with Regard to TheirAffinity to Different Target Molecules in Native Materials bySimultaneously Determining the Unbound Portions of Two Different Markers

5.1. Conception

SCH23390 and spiroperidol possess high affinity to D₁- and D₂-typereceptors in the central nervous system, and are used as markers for therespective target molecules. As native material which contains both D₁and D₂ receptors in sufficient amount, a membrane fraction from thestriatum of the porcine brain is employed. Test substances aresimultaneously examined in a binding assay with respect to their abilityto inhibit the specific binding of SCH23390 to D₁ receptors or thespecific binding of spiroperidol to D₂ receptors, respectively.

5.2. Recovery of the Receptor Material

The striata of 20 porcine brains (raw mass 20 g) were homogenized with aPotter in the ten-fold volume of 0.32 mol/L of sucrose, and werecentrifuged at 1,000 g for 10 min at 4° C. The supernatant wascentrifuged at 20,000 g for 10 min at 4° C. The pellets were resuspendedin 50 mmol/L of Tris HCl, pH 7.4 (buffer), and centrifuged at 30,000 gfor 10 min at 4° C. The last step was repeated. The resultant membranefraction was frozen in liquid N₂.

5.3. Binding Assays

In a first binding assay (B1), an aliquot of the membrane fraction whichcontained 1.7 mg of protein was incubated with 20 nmol/L of each ofSCH23390 and spiroperidol in 50 mmol/L of Tris HCl, pH 7.4 in a totalvolume of 250 μL for 40 min at 25° C. in a 1.5 ml PP reaction vessel.Subsequently, the membrane particles were centrifuged off at 50,000×gfor 20 min at 4° C. 200 μL of the supernatant (Ü1) were taken off.

A second binding assay (B2) which additionally contained butaclamol at aconcentration of 100 μmol/L was carried out analogously to B1 andafforded Ü2. A third binding assay (B3) which additionally containedhaloperidol as a test substance at a concentration of 1 μmol/L wascarried out analogously to B1 and afforded Ü3. A fourth binding assay(B4) which additionally contained chlorpromazine as a test substance ata concentration of 1 μmol/L was carried out analogously to B1 andafforded Ü4. A fifth binding assay (B5) which additionally containedmethyl benzoate as a test substance at a concentration of 1 μmol/L wascarried out analogously to B1 and afforded Ü5. All binding assays B1 toB5 were carried out as double determinations.

A further supernatant Ü0 which contained neither SCH23390 norspiroperidol during the incubation was recovered in analogous manner.

5.4. Quantification of the Marker

The mass spectroscopic determination of the marker was carried out on anApplied Biosystems API 2000 with TurbolonSpray source in the MRM mode,to which a Hewlett Packard HP1100 LC system was coupled. The markersSCH23390 and spiroperidol were measured directly side by side in thesupernatants Ü1–Ü5 by means of the MRM method which was based on themass transitions of 288.1/91.1 for SCH23390 and 395.9/122.9 forspiroperidol, respectively, and were quantified in comparison toexternal standards (1 nmol/L to 5 nmol/L of SCH23390 and spiroperidol inÜ0). All samples were measured twice.

-   Column: Superspher 60 RP select B4 μm 125×2 mm with 10×2 mm    pre-column-   Eluent: 0.1% HCOOH in water/acetonitrile (65/35)-   Flow: 0.35 ml/minute (isocratic)-   Sample volume: 20 μL (injected by means of Autosampler)    5.5. Analysis

In Ü1–Ü5 the following concentrations of unbound markers were determined(average values of doubles and double LCMS determination):

SCH23390 Spiroperidol Ü1: 2.88 nmol/L 1.93 nmol/L Ü2: 4.15 nmol/L 2.75nmol/L Ü3: 2.77 nmol/L 2.78 nmol/L Ü4: 2.71 nmol/L 2.29 nmol/L Ü5: 2.71nmol/L 1.89 nmol/L

Each of the differences of the markers in Ü1 and Ü2 describes thespecific binding for SCH23390 and spiroperidol (for SCH23390 1.27nmol/L; for spiroperidol 0.82 mnol/L).

Among the studied test substances (haloperidol, chlorpromazine andmethyl benzoate), none is capable, at the employed concentration of 1μmol/L, to inhibit the specific binding of the marker SCH23390 toD₁-type receptors. Apparently, none of the studied test substancesexhibits (in the concentration employed) affinity to D₁-type receptors.

In the employed concentration of 1 μmol/L, methyl benzoate is notcapable of inhibiting the specific binding of spiroperidol to D₂-typereceptors. Apparently, methyl benzoate exhibits (in the concentrationemployed) no affinity to D₂-type receptors. Haloperidol completelyinhibits the specific binding of the marker spiroperidol to D₂-typereceptors, while chlorpromazine inhibits the specific binding of themarker spiroperidol to D₂-type receptors only at about 50%. Apparently,haloperidol has a higher affinity to D₂-type receptors thanchlorpromazine.

1. A method for determining the binding behavior of ligands which bindspecifically to target molecules at at least one binding site, whichmethod comprises: (A) preparing a first mixed phase wherein the ligandsare contacted with (i) markers which bind specifically to the targetmolecules, and (ii) with the target molecules, wherein the ligands arepresent in a concentration K1, the markers are present in aconcentration K2 and the target molecules are present in a concentrationK3; (B) preparing a second mixed phase wherein the markers are contactedwith the target molecules; (C) incubating the first and second mixedphases under identical conditions, which conditions permit a binding ofthe ligands and the markers to the target molecules, resulting in (a) aquantity M1 of markers GM1 that are bound to target molecules and aconcentration K4 of unbound markers in the first mixed phase and (b) aquantity M2 of markers GM2 that are bound to target molecules and aconcentration K5 of unbound markers in the second mixed phase; (D)separating the markers GM1 from the first mixed phase and separating themarkers GM2 from the second mixed phase; (E1) determination of theconcentrations K4 and KS, and calculating a concentration K6 from K5,wherein K6 is a concentration of unbound markers in the second mixedphase under the assumption that the markers have been contacted thereinin the concentration K2 with the target molecules in the concentrationK3, and determining the binding behavior of the ligands from the ratioof the concentrations K6 and K4, or (E2) determination of the quantitiesM1 and M2, and calculating a quantity M3 from M2, wherein M3 is aquantity of bound markers in the second mixed phase under the assumptionthat the markers have been contacted therein in the concentration K2with the target molecules in the concentration K3, and determining thebinding behavior of the ligands from the ratio of the quantities M3 andM1 ; wherein the markers are present in a native form and thedetermination of K4 and K5 or M1 and M2 is carried out by massspectrometry.
 2. The method of claim 1, wherein in (B) the markers arepresent in a concentration K2 and the target molecules are present in aconcentration K3.
 3. The method of claim 1, wherein the markers GM1comprise specifically bound markers SGM1 and the markers GM2 comprisespecifically bound markers SGM2 and wherein SGM1 and SGM2 are liberatedafter (D), and quantities thereof are used in (E2) instead of M1 and M2.4. The method of claim 1, further comprising repeating (A) to (E1) or(A) to (E2) wherein in at least one of (A) and (B) an agent whichcompetes with the markers for a specific binding to the target moleculesis added to at least one of the first and second mixed phases in anamount which substantially prevents or cancels a specific binding of themarkers to the target molecules.
 5. The method claim 1, furthercomprising repeating (A) to (E1) or (A) to (E2) at least once with adifferent ratio of K1 and K3.
 6. The method of claim 1, wherein (D)comprises at least one of dialysis, precipitation, adsorption, bindingto immobilized molecules which have an affinity to at least one of thetarget molecules, complexes of the target molecules and the markers andcomplexes of the target molecules and the ligands, centrifugation,chromatography, and filtration.
 7. The method of claim 1, wherein (D)comprises ultrafiltration.
 8. The method of claim 1 , wherein (D) iscarried out under conditions under which at least one of(a) theconcentrations of the unbound markers in the first and the second mixedphases and (b) the amounts of the bound markers GM 1 and GM2 remainconstant.
 9. The method of claim 1, wherein the mass spectrometry iscarried out in one of the MS, MS/MS and MS^(n) modes.
 10. The method ofclaim 9, wherein the mass spectrometry is preceded by at least one of acapillary electrophoresis, a gas chromatography and a liquidchromatography.
 11. The method of claim 1, wherein the mass spectrometryis preceded by a HPLC.
 12. The method of claim 11, wherein the HPLCcomprises reverse phase chromatography.
 13. The method of claim 1,wherein prior to the mass spectrometry a sample preparation by means ofat least one of extraction, filtration, gas chromatography, capillaryelectrophoresis, liquid chromatography, fast preformance liquidchromatography, reverse phase chromatography, size exclusionchromatography and affinity chromatography is carried out.
 14. Themethod of claim 1, wherein prior to the mass spectrometry a samplepreparation by means of at least one of solid phase extraction,liquid-liquid extraction, ultrafiltration and HPLC is carried out. 15.The method of claim 1, wherein the first and the second mixed phases of(A) and (B) comprise at least one of more than 10 different targetmolecules and more than 10 different markers.
 16. The method of claim15, wherein the first and the second mixed phases of (A) and (B)comprise at least one of more than 100 different target molecules andmore than 100 different markers.
 17. The method of claim 1, wherein thefirst and the second mixed phases of (A) and (B) comprise a commonmarker which is specific to different target molecules.
 18. The methodof claim 15, wherein the different markers differ from each other withrespect to at least one of the molar mass, the formation of ions ordaughter ions in the mass spectrometry, the chromatographic behavior andthe electrophoretic behavior.
 19. The method of claim 1, wherein thefirst mixed phase of (A) comprises at least one of more than 10different target molecules and more than 10 different markers.
 20. Themethod of claim 19, wherein the first mixed phase of (A) comprises atleast one of more than 100 different target molecules and more than 100different markers.
 21. The method of claim 19, wherein the ligands aregenerated by methods of combinatorial chemistry.
 22. The method of claim1, further comprising at least one of identifying ligands which arebound by the target molecules and determining a quantity of ligandswhich are bound by the target molecules.
 23. The method of claim 22,wherein mass spectrometry is used for the at least one of identifyingthe bound ligands and determining the quantity thereof.
 24. The methodof claim 23, wherein prior to mass spectrometry the bound ligands arespecifically liberated from the target molecules.
 25. The method ofclaim 1, wherein the markers are used as external standard for thedetermination of K4 and KS or M1 and M2.
 26. The method of claim 1,wherein after (D) at least one marker which comprises a markingsubstance is added as an internal standard to at least one the first andsecond mixed phases.
 27. The method of claim 1, wherein the targetmolecules comprise target molecules that are embedded in membranestructures.
 28. The method of claim 1, wherein the target moleculescomprise target molecules that are not bound to a solid support.
 29. Themethod of claim 1, further comprising a regeneration of the targetmolecules after one of (D) and (F2).
 30. The method of claim 1, whereinat least one of the target molecules, markers and ligands are selectedfrom peptides, proteins, hormones, nucleic acids, sugars, polymers, lowmolecular weight compounds of natural or synthetic origin and structureson or inside cells, cell fragments, cell homogenates, synaptosomes,liposomes, vesicles, tissue cuts, viruses, components and fragmentsthereof, capsides, components and fragments thereof, and mixtures ofnatural substances.
 31. The method of claim 30, wherein at least one ofthe target molecules, markers and ligands are selected from receptors,prions, enzymes, transport proteins, ion channels, antibodies, synapticplasma membrane vesicles and vesicles found inside cells.
 32. A processfor determining the capability of a chemical species to exhibitpharmaceutical activity, wherein the process comprises carrying out themethod of claim
 1. 33. The process of claim 32, wherein the ligandscomprise the chemical species.